WO2023042156A1 - Procédés et appareil pour tester des médicaments et des produits biologiques pour l'immuno-oncologie à l'aide de microorganosphères - Google Patents

Procédés et appareil pour tester des médicaments et des produits biologiques pour l'immuno-oncologie à l'aide de microorganosphères Download PDF

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WO2023042156A1
WO2023042156A1 PCT/IB2022/058785 IB2022058785W WO2023042156A1 WO 2023042156 A1 WO2023042156 A1 WO 2023042156A1 IB 2022058785 W IB2022058785 W IB 2022058785W WO 2023042156 A1 WO2023042156 A1 WO 2023042156A1
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moss
cells
tissue
patient
drug
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PCT/IB2022/058785
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English (en)
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Xiling Shen
Shengli DINGL
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Xilis, Inc
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Publication of WO2023042156A1 publication Critical patent/WO2023042156A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0693—Tumour cells; Cancer cells
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5082—Supracellular entities, e.g. tissue, organisms
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • Multicellular tumor spheroids were first described in the early 70s and obtained by culture of cancer cell lines under non-adherent conditions. Spheroids are typically formed from cancer cell lines as freely floating cell aggregates in ultra-low attachment plates. Spheroids have been shown to maintain more stem cell associated properties than 2D cell culture.
  • the dissociated cells may be modified by treatment with one or more agents.
  • the cells may be genetically modified.
  • the cells may be modified using CRISPR-Cas9 or other genetic editing techniques.
  • the cells may be transfected by any appropriate method (e.g., electroporation, cell squeezing, nanoparticle injection, magnetofection, chemical transfection, viral transfection, etc.), including transfection with of plasmids, RNA, siRNA, etc. Alternatively, the cells may be used without modification.
  • a method e.g., of forming a plurality of MOSs, may include combining a dissociated tissue sample and a fluid matrix material to form an unpolymerized mixture; forming a plurality of droplets from a continuous stream of the unpolymerized mixture wherein the droplets have less than a 25% embodiment in size; and polymerizing the droplets by warming to form a plurality of MOSs each having between 1 and 200 dissociated cells distributed within each MOS.
  • any assay may be used.
  • genomic, transcriptomic, proteomics, or meta-genomic markers such as methylation
  • meta-genomic markers such as methylation
  • any of these compositions and methods described herein may be used to identify or examine one or more markers and biological/physiological pathways, including, for example, exosomes, which may assist in identifying drugs and/or therapies for patient treatment.
  • the plurality of MOSs may be cryopreserved or assayed before six passages, whereby heterogeneity of the cells within the MOSs is maintained. Any of these methods may further include modifying the cells within the dissociated tissue sample prior to forming the droplets.
  • compositions of matter comprising a plurality of cryopreserved MOSs, wherein each MOS has a spherical shape having a diameter of between 50 pm and 500 pm, wherein the MOSs have less than a 25% embodiment in size, and wherein each MOS comprises a polymerized base material, and between about 1 and 500 dissociated primary cells distributed within the base material that have been passaged less than six times, whereby heterogeneity of the cells within the MOSs is maintained.
  • the MOSs include immune cells from the tissue of origin.
  • the primary cells may be primary tumor cells.
  • the dissociated primary cells may have been genetically or biochemically modified.
  • the plurality of cryopreserved MOSs may have a uniform size with less than 25% variation in size.
  • the plurality of cryopreserved MOSs may comprise MOSs from various sources. In any of these MOSs, the majority of cells in each MOS may comprise cells that are not stem cells.
  • the primary cells comprise metastatic tumor cells.
  • the primary cells may comprise both cancer cells and stroma cells.
  • the primary cells comprise tumor cells and one or more of: mesenchymal cells, endothelial cells, and immune cells.
  • the MOSs described herein may include any appropriate number of primary tissue cells initially in each MOS, for example less than about 200 primary cells, or more preferably less than about 150 primary cells, or more preferably less than about 100 primary cells, or more preferably less than about 75 primary cells, or less than about 50 cells, or less than about 30 cells, or less than about 25 cells, or less than about 20 cells or less than about 10 cell, or less than about 5 cells, etc.).
  • methods of operating a MOS forming apparatus comprising: receiving an unpolymerized mixture comprising a chilled mixture of a dissociated tissue sample and a first fluid matrix material in a first port; receiving a second fluid that is immiscible with the unpolymerized mixture in a second port; combining a stream of the unpolymerized mixture with one or more streams of the second fluid to form droplets of the unpolymerized mixture having a uniform size that varies by less than 25%; and polymerizing the droplets of the unpolymerized mixture to form a plurality of MOSs.
  • any of these methods may include coupling a first reservoir containing the unpolymerized mixture in fluid communication with the first port.
  • the method may include combining the dissociated tissue sample and the first fluid matrix material to form the unpolymerized mixture.
  • the method includes adding the unpolymerized mixture to a first reservoir in fluid communication with the first port.
  • These methods may include coupling a second reservoir containing the second fluid in fluid communication with the second port. Any of these methods may include adding the second fluid to a second reservoir in fluid communication with the second port.
  • receiving the second fluid comprises receiving an oil.
  • these methods may include determining that the tumor is still responding to the drug formulation after one or more administrations of the drug to the patient by receiving a second patient biopsy after the patient has been treated with the drug formulation and forming a second plurality of MOSs from the second patient biopsy, exposing at least some of the second plurality of MOSs to the drug formulation, and measuring the effect of the drug formulation on cells within the at least some of the second plurality of MOSs.
  • any of these methods may include biopsying the patient to collect the patient biopsy (or otherwise taking a tissue sample from a patient or a sample of patient-derived tissues or cells) and/or treating the patient with the drug formulation, or assisting a physician in treating the patient (e.g., advising the physician as to which drug formulations would be effective).
  • the time between receiving the biopsy and reporting may be less than about 21 days (e.g., less than about 15 days, less than about 14 days, less than about 13 days, less than about 12 days, less than about 11 days, less than about 10 days, less than about 9 days, less than about 8 days, less than about 7 days, etc.).
  • MOSs can be used for testing certain therapies that were previously difficult to test.
  • cells of the immune system (“immune cells”) present in the biopsied patient derived tissues (e.g., from a tumor) may also be present and persist in MOSs upon their formation, even after the extensive processing for MOS formation described herein.
  • Immune cells in MOS prepared as described herein can persist for 7 days or more, and in some cases 14 days or more. In some embodiments immune cells may persist for 21 days or longer.
  • MOS formation as described herein allows for immune cells from the patient derived tissues to be incorporated, the accuracy of testing the aforementioned drug formulations in MOSs is superior to testing in traditional bulk organoids, and the interactions between immune cells and tumor cells can be monitored. These interactions may confer useful information about the effect of a drug on the interface and/or interaction between immune cells and cancer cells.
  • immune cells derived from a patient may be separately introduced into MOS that have already formed, because of ease of penetration.
  • Patient derived tissues e.g., from a tumor
  • alternative methods of demulsification are used, for example, a demulsifying device that may make incorporate (i) magnetic separation or (ii) laminar flow properties and small microstructures to allow for gentle filtering of oil. Such alternative methods can also be utilized to achieve persistence of the immune cells in the MOS.
  • FIGS. 4A to 4E illustrate examples of Patient-Derived MOSs formed as described herein to include ten dissociated primary tissue cells per MOS.
  • FIG. 4A shows the MOSs shortly after formation (at low magnification).
  • FIG. 4B shows a higher magnification view of some of the MOSs of FIG. 4A taken after culturing for two days.
  • FIG. 4C shows the MOSs after culturing for three days.
  • FIG. 4D shows the MOSs after culturing for four days.
  • FIG. 4E shows the MOSs after culturing for five days.
  • FIGS. 12A to 12B show a plurality of Patient-Derived MOSs following separation from the immiscible fluid within a few hours of formation of the Patient- Derived MOSs at low magnification (FIG. 12A) and higher magnification (FIG. 12B).
  • FIG. 13 is another example of an image showing a plurality of Patient- Derived MOSs formed as described herein.
  • FIG. 14 is a chart illustrating the size distribution of the diameters from a plurality of Patient-Derived MOSs formed from an exemplary biopsy sample.
  • FIGS. 16A to 16B is another example, similar to that shown in FIGS. 15A- 15B, showing low and higher magnification views, respectively, of one example of a plurality of Patient- Derived MOSs.
  • FIG. 16A is an unstained image, while in FIG. 16B the MOSs have been stained with Trypan blue (arrows) to show that the dissociated cells in the MOSs indicated that the cell remail viable (e.g., living) within the MOS.
  • FIGS. 17A-17E illustrates one example of a method of assaying a plurality of Patient- Derived MOSs formed from a patient tumor biopsy, to determine a drugresponse profile to multiple drug formulations. The illustrated procedure takes less than two weeks (e.g., approximately one week) from biopsy to results.
  • FIG. 19 schematically illustrates an example of a method for treating a patient including multiple iterations of rapidly forming and assaying a plurality of Patient- Derived MOSs as part of the treatment procedure.
  • FIG. 21 schematically illustrates a method of operating an apparatus for forming a plurality of Patient-Derived MOSs similar to that shown in FIG. 20.
  • FIGS. 22A to 22D illustrate one example of a validation of a method of using a plurality of Patient-Derived MOSs as described herein to identify drug resistance.
  • FIG. 22A illustrates the use of traditional (“2D”) tumor cell assay methods for predicting drug resistance.
  • FIG. 22B illustrates the use of one example of a Patient- Derived MOS method as described herein, to assay for drug resistance for predicting drug sensitivity.
  • FIGS. 22C and 22D show that the Patient-Derived MOS based method accurately predicted the actual response of the tumor (drug responsive), unlike traditional cultured cells.
  • FIGS. 23A to 23D illustrate another example validating the use of Patient- Derived MOSs as described herein to identify drug resistance, showing the predicted drug response to both Oxaliplatin and Irinotecan as consistent with actual tumor response following treatment with these drugs.
  • FIGS. 27A to 27C illustrate examples of human liver MOSs formed from human liver tissue.
  • FIG. 27 A shows the MOSs at day 1 , seeded with 40 cells/droplet.
  • FIGS. 27B and FIG. 27C show the MOSs at day 18.
  • the MOSs are hepatocyte-like structures, while FIG. 27C shows Cholangiocyte-like MOSs.
  • FIGS. 33A and 33B show examples of toxicity assays using mouse liver MOSs.
  • FIG. 33A shows that the sizes of the tissue in the mouse liver MOSs in the control group are relatively large (as indicated by the arrows).
  • FIG. 33 A showing the acetaminophen (10 mM) treatment group, the tissue in most of the MOSs is smaller and contains many dead cells.
  • FIG. 35C illustrates the use of a chemical demulsification agent, in this case PFO (10% PFO) used to remove oil from the gel droplets.
  • FIG. 35D illustrates gel droplets that have been processed to remove the oil using a porous hydrophobic surface (e.g., membrane).
  • FIG. 37A through 37C show illustrative embodiments of a demulsifier.
  • FIG. 37A shows a schematic diagram of a first illustrative embodiment of a demulsifier.
  • FIG. 37B shows a schematic diagram of a second illustrative embodiment of a demulsifier.
  • Fig. 37C shows cross-sectional view of a third illustrative embodiment of a demulsifier.
  • FIG. 49 illustrates the effect of combining the treatment of Nivolumab with TILs.
  • the unpolymerized mixture may then be dispensed as droplets, e.g., into an immiscible material, such as an oil, in a manner that controls the formation of the size of the droplets and therefore the size of the MOSs formed 607.
  • an immiscible material such as an oil
  • uniformly-sized droplets may be formed by combining a stream of the unpolymerized material into one or more (e.g., two converging) streams of the immiscible material (e.g., oil) so that the flow rates and/or pressures of the two streams may determine how droplets of the unpolymerized material are formed as they intersect the immiscible material.
  • the droplets may be polymerized 609 to form the MOSs in the immiscible material.
  • the immiscible material may be heated or warmed to a temperature that causes the unpolymerized mixture (e.g., the fluid matrix material in the unpolymerized material) to polymerize.
  • the MOSs may be separated from the immiscible fluid, e.g., the MOSs may be washed to remove the immiscible fluid 611 , and placed in a culture media to allow the cells within the MOSs to grow.
  • the MOSs may be cultured for any desired time, or may be cryopreserved and/or assayed immediately.
  • the droplets may be formed by other methods that may allow for the size of the droplet to be controlled as described herein.
  • the droplets may be formed by printing (e.g., by printing droplets onto a surface). This may reduce or eliminate the need for an additional recovery step of emulsification/de-emulsification.
  • the droplets may be printed onto a surface, such as a flat or shaped surface, and polymerized.
  • the method for forming the MOSs may be automated, or performed using one or more apparatuses.
  • the method of forming the MOSs may be performed by an apparatus that allows the selection and/or control of the size of the MOSs (and therefore the density of the number of cells).
  • FIG. 7A illustrates one example of an apparatus 700 for forming MOSs as described.
  • the entire apparatus 700 may be enclosed in a housing 702 or a portion of the apparatus 704 may be enclosed in a housing.
  • the housing may include one or more openings or access portions on the device, e.g., for adding the immiscible fluid and/or the unpolymerized mixture.
  • the input from the immiscible fluid channel(s) may be at an angle relative to the angle (and point of intersection) with the unpolymerized material.
  • FIG. 7C as in all figures in this description showing dimensions, the dimensions shown are exemplary only, and are not intended to be limiting, unless they otherwise specify.
  • the junction region 937 is shaped as described above, so that the channel carrying the unpolymerized mixture 911 intersects one or more (e.g., two) channels 909 carrying a fluid, such as an oil, that is immiscible with the unpolymerized mixture.
  • a fluid such as an oil
  • the unpolymerized mixture is pressurized to flow at first rate out of the first channel 911 , the flowing immiscible fluid in the intersecting channels, 909, 909’, permit a predefined amount of the unpolymerized mixture to pass before pinching it off to form a droplet 903 that is passed into the outlet channel 939.
  • a minced (e.g., dissociated) clinical (e.g., biopsy or resected) sample of tissue may be is mixed with a temperaturesensitive gel (i.e. MATRIGEL, at 4 degrees C) to form the unpolymerized mixture.
  • a temperaturesensitive gel i.e. MATRIGEL, at 4 degrees C
  • This unpolymerized mixture may be placed into the microfluidic device that may generate droplets (e.g., water-in-oil droplets) that are uniform in volume and material composition.
  • the dissociated tumor cells may be partitioned into these droplets.
  • the gel in the unpolymerized material may solidify upon heating (e.g., at 37 degrees C), and the resulting MOSs may be formed.
  • the junction is shown as a T- or X-junction in which the flow focusing of the microfluidics forms the controllable size of the MOS.
  • the droplets may be formed by robotic micro-pipetting, e.g., into an immiscible fluid and/or onto a solid or gel substrate.
  • the droplets of unpolymerized material may be formed in the requisite dimensions and reproducibility by micro-capillary generation.
  • FIGS. 11A and 11 B show examples of MOSs in oil formed as described above.
  • the cells within these MOSs derived from a single biopsy sample, are viable, as seen by vital dye staining, as shown in FIGS. 15A-15B and 16A-16B.
  • FIG. 12A-12B illustrates MOSs having tumor cells (similar to those shown in FIG. 11 A-11 B) that may be washed to remove the immiscible material (e.g., oil). This immiscible material may be removed relatively quickly after forming the MOS in order to prevent harm to the cells within the MOS.
  • immiscible material e.g., oil
  • membrane-based demulsification (e.g., using a hydrophobic membrane to remove oil from MOSs) may be utilized to improve persistence of the immune cells in the MOSs.
  • FIG. 35A shows an example of gel droplets in oil.
  • the gel droplets may be used for an assay immediately, cultured, and/or stored (e.g., by cryopreservation).
  • the viability, particularly in culture is negatively affected by including oil with the gel droplets.
  • the presence of oil may make it difficult or impossible to accurately assay and/or manipulate the gel droplets.
  • the gel droplets within (or including some) oil may clump or cluster together, preventing isolation and manipulation of individual gel droplets. Thus, it is desirable to remove the oil.
  • FIGS. 35B and 35C illustrate examples of methods that may be used for removing oil from the gel droplets; these techniques are less thorough and effective, and may in fact be more complicated, than the methods also described herein using a porous hydrophobic surface.
  • FIG. 35B illustrates the gel droplets for which an antistatic gun was used to remove oil.
  • the oil was not completely removed, and further, the resulting gel droplets (shown here by asterisks) are left with oil and debris, possibly resulting from destruction of some gel droplets during the demulsification step.
  • FIG. 35B illustrates the gel droplets for which an antistatic gun was used to remove oil.
  • the oil was not completely removed, and further, the resulting gel droplets (shown here by asterisks) are left with oil and debris, possibly resulting from destruction of some gel droplets during the demulsification step.
  • FIG. 35B illustrates the gel droplets for which an antistatic gun was used to remove oil.
  • FIG. 36 illustrates one example of a general method of forming/and or processing (including removing oil) gel droplets using a porous hydrophobic surface.
  • the method may optionally begin with a primary tissue sample (or other source of cells to be included in the gel droplets); the tissue sample may be dissociated and/or suspended 501.
  • the cells may be modified 503.
  • the dissociated cells may then be combined with an unpolymerized matrix material 505, and streamed into an oil to form the gel droplets within the oil; the matrix material with the combined dissociated cells (and any additional components) may then be polymerized, as described above, e.g., by increasing the temperature.
  • These steps may generally be part of a step or multiple steps for forming the gel droplets 507, and all or some of these steps may be automated, e.g., by an apparatus.
  • FIG. 37A is a schematic diagram of a demulsifier 250 coupled to a polymerizer 240 and an output 252.
  • the demulsifier 250 may be coupled to an output of the polymerizer 240 and the output 252 may be coupled to an output of the demulsifier 250.
  • the polymerizer 240 and demulsifier 250 may be temperature regulated at about 37 °C.
  • FIG. 37B is a schematic diagram of a demulsifier 600 based on magnetic separation.
  • the demulsifier 600 may comprise a first inlet 610A (e.g., oil and micro-organosphere inlet), first outlet 612A (e.g., oil and waste outlet), second inlet 620A (e.g., growth media and wash inlet), and second outlet 622A (e.g., growth media and micro-organosphere outlet).
  • the first inlet 610A and the second inlet 620A may be disposed on a first side of the demulsifier 600 and the first outlet 612A and the second outlet 622A may be disposed on a second side of the demulsifier 600 opposite the first side.
  • a mixture of a first fluid (e.g., oil) and polymerized micro- organospheres 650 may be received in first inlet 610A.
  • a second fluid e.g., growth media, wash fluid, aqueous solution
  • the demulsifier 600 may be configured for laminar flow, as shown in FIG. 6A, such that the hydrophobic properties of the aqueous fluid from the second inlet 620A and oil from first inlet 610A do not mix within the demulsifier 600.
  • first flow stream 630A e.g., oil flow stream
  • second flow stream 632A e.g., aqueous flow stream
  • the demulsifier 600 may further comprise a magnet 640 that serves as a flow separator configured to separate the micro- organospheres 650 that contain magnetic nanoparticles from the first flow stream 630A (e.g., oil flow stream).
  • the magnet 640 may be configured to extend along a length of the demulsifier 600.
  • the magnet 640 may separate the micro-organospheres 650 from the first flow stream 630A (e.g., an oil flow stream) and into the second flow stream 632A (e.g., an aqueous flow stream).
  • first flow stream 630A e.g., an oil flow stream
  • second flow stream 632A e.g., an aqueous flow stream
  • FIG. 37C is a demulsifier 602 that takes advantage of laminar flow properties and small microstructures (e.g., micro-pillars) to filter polymerized droplets from oil into media.
  • the demulsifier 602 may comprise a first inlet 610B (e.g., oil and micro-organosphere inlet), first outlet 612B (e.g., oil and waste outlet), second inlet 620B (e.g., growth media and wash inlet), and second outlet 622B (e.g., growth media and micro-organosphere outlet).
  • first inlet 610B e.g., oil and micro-organosphere inlet
  • first outlet 612B e.g., oil and waste outlet
  • second inlet 620B e.g., growth media and wash inlet
  • second outlet 622B e.g., growth media and micro-organosphere outlet
  • the first inlet 610B and the second inlet 620B may be disposed on a first side of the demulsifier 602 and the first outlet 612B and the second outlet 622B may be disposed on a second side of the demulsifier 602 opposite the first side.
  • a mixture of a first fluid (e.g., oil) and polymerized micro- organospheres 650 may be received in first inlet 610B.
  • a second fluid e.g., growth media, wash fluid, aqueous solution
  • the demulsifier 602 may be configured for laminar flow, as shown in FIG.
  • the demulsifier 602 may comprise a flow separator 660 (e.g., a set of micro-pillars) configured to separate micro-organospheres 650 from the first flow stream (e.g., oil flow stream).
  • the flow separator 660 may be configured to extend along a length of the demulsifier 602.
  • the micro-pillars of the flow separator 640 may separate the micro-organospheres 650 from the first flow stream (e.g., oil flow stream) and into the second flow stream (e.g., aqueous flow stream).
  • the micro-pillars can be positioned at an angle of about one degree from the first flow stream (e.g., oil flow stream) into the second flow stream (e.g., aqueous flow stream), thereby forcing the micro- organospheres 650 from the first flow stream into the second flow stream while allowing each flow stream to remain flowing in parallel.
  • the spacing of the micro-pillars is such that the micro organospheres 650 are unable to pass through the micro-pillars and the spacing can be varied in fabrication depending on the expected droplet size.
  • MOSs described herein provide a good model for the effectiveness of various drug formations. A variety of drugs can be used and interacted with MOSs.
  • Exemplary drugs include (but are not limited to) MAPK inhibitors (e.g., Vemurafenib, Dabrafenib, PLX8349, Cobimetinib, Trametinib, Selumetinib, and BVD-523), checkpoint inhibitors (e.g., T-cell targeted immunomodulators, Pembrolizumab, Avelumab, Durvalumab, Ipilimumab, TSR-022, MGB453, BMS-986016, and LAG525), other immunomodulators (e.g., anti-CD47 antibodies, and ADCC therapies), apoptosis inhibitors (e.g., ABT-737, WEHI-539, ABT- 199) potential contributing pathways (e.g., Afuresetib, Idasanutlin, and Infliximab), chemotherapy agents (e.g., Cytarabine), cell therapy, cancer vaccine, oncolytic viruses, and bi-specific antibodies.
  • MAPK inhibitors
  • FIGS. 43A and 43B shows the efficacy of Nivolumab on MOSs derived from pulmonary and renal tumor biopsies. Assays of the MOSs for these pulmonary and renal tumor biopsies were prepared with an Annexin V marker to indicate cellular apoptosis. As can be seen in FIGS. 43A and 43B, the MOSs display a good response to Nivolumab (in the form of tumor cell apoptosis). If this test were run on, for example, traditional bulk organoids, the results would not be meaningful (due to the lack of patient immune cells) leading to uncertainty in the best course of action for patient treatment.
  • FIGS. 44A and 44B show the efficacy of Lenalidomide and Bortezoid on MOSs derived from multiple myeloma (MM) bone marrow biopsies. MM MOSs (on day 11) were treated with Lenalidomine (5uM) or Bortezoid (2nM). Caspase 3/7 green dye was added in the assay to monitor apoptosis. Incucyte images were taken every 2 hours for 4 days. As can be seen in FIGS. 44A and
  • FIG. 45 shows the efficacy of ESK1 (a T-cell receptor-mimic antibody) on MOSs derived from a pulmonary Biopsy. Assays of the MOSs for this pulmonary tumor biopsy were prepared with an Annexin V marker to indicate cellular apoptosis. As can be seen in FIG 45, the MOSs display a good response to ESK1 (in the form of tumor cell apoptosis). If this test were run on, for example, traditional bulk organoids, the results would be less accurate (because ESK1 would not be able to reach its target) leading to uncertainty in the best course of action for patient treatment.
  • ESK1 a T-cell receptor-mimic antibody
  • MOSs will readily uptake infused immune cells to provide a good model for the effectiveness of various immune cell therapies.
  • the MOSs described herein are highly uniform in diameter, and may have a very low size, e.g., diameter, variance. This is illustrated, for example, in FIG. 14, showing a distribution of one example of droplet diameter sizes.
  • MOSs survive in a biologically significant manner, allowing them to provide clinically and physiologically relevant data, particularly with respect to drug response, as will be described in FIGS. 22A-22D and 23A-23D.
  • the MOSs described herein permit tissue extract/biopsy originated cells to grow exceptionally well and provide more representative data, especially as compared to organoids or spheroids. Without being bound by a particular theory, this may be because the cells may have a more constrained cell density in the MOSs, permitting cells to communicate without inhibiting each other while sharing signals.
  • the MOSs also have a very large surface to volume ratio, more readily permitting transmission of growth factors and other signals to penetrate into the MOSs (e.g., the MOSs are less diffusion limited).
  • the screening assay may be automated. This may enable repeatable and automated workflow, which may increase the number of drugs screened from a few to hundreds.
  • FIGS. 17A-17E illustrate one example of this workflow.
  • a tumor biopsy is taken and a plurality (e.g., >10,000) MOSs are formed as described above (in FIG. 17A the junction region forming the MOSs is illustrated). Thereafter, the MOSs may be recovered and washed (e.g., to remove the immiscible (e.g., oil) material in which they were formed). The MOSs may then be plated into one or more microwell plates. As shown in FIG.
  • the workflow shown in FIGS. 17A-17E may enable an integrated device to be used for growing, dosing and/or reviewing the MOSs.
  • freshly biopsied or resected patient tumor samples may be disassociated and seeded into gel with regents to form the MOSs (as described above).
  • a portion of the MOSs formed may be cryopreserved. The rest may be recovered and incubated until seeded into microwell plates for drug testing or screening as just described.
  • Growth and viability assays may be performed on the MOSs, which may be imaged and tracked. Their response to drug treatments, such as IC-50, cytotoxicity, and growth curves, may be measured to identify effective therapeutics against the patient’s tumor.
  • the use of the MOSs described herein for screening may be automated or manually performed.
  • Virtually any screening technique may be used, including imaging by one or more of: confocal microscopy, fluorescent microscopy, liquid lens, holography, sonar, bright and dark field imaging, laser, planar laser sheet, including high-throughput embodiments of image-based analysis methods (e.g., using computer vision, and/or supervised or unsupervised model, e.g., CNN).
  • Downstream screening may include sampling the culture media and/or performing genetic or protein screening (e.g., scRNA-seq, ATAC-seq, proteomics, etc.) on cells from the MOSs.
  • genetic or protein screening e.g., scRNA-seq, ATAC-seq, proteomics, etc.
  • FIGS. 20 and 21 illustrate another example of an apparatus for forming a plurality of MOSs as described herein.
  • the apparatus may include a plurality of MOS forming junctions, in which the immiscible material (e.g., oil) 2002 may be added to a reservoir and/or port 2004 in the device.
  • the unpolymerized material 2006 in this example, including the dissociated biopsy cells and the fluid matrix material
  • a second or additional material e.g., a biologically active agent
  • FIG. 21 illustrates the method of forming the MOSs using an apparatus as shown in FIG. 20.
  • the resulting MOSs include both the target (e.g., tumor) biopsy cells and also one or more additional biologically active agents that are combined to form the MOS.
  • a first channel 2103 may include the unpolymerized material (including the dissociated biopsy cells and the matrix material)
  • a second channel 2107 includes an additional active biological material
  • a pair of intersecting channels 2109, 2109’ carrying the immiscible material e.g., oil
  • FIG. 22B For comparison a plurality of MOSs were generated from a patient biopsy, as shown in FIG. 22B.
  • the MOSs showed significant decreases in cell survival from the tumor MOSs, predicting drug sensitivity.
  • the tumor responded to treatment, as shown in FIG. 22C (before treatment) and 22D (post treatment).
  • MOSs may be formed from biopsied renal tissue.
  • instruments used may include: a tube rotator or 100 pm and 70 pm cell strainer, 15 mL conical tubes, 50 mL conical tubes, Razor blades, Tweezers and surgical scissors, Petri dish (100 x 15 mm) or tissue culture dish.
  • the reagents may include: EBM-2 media, Collagenase (5 mg/mL stock), Hank’s Balanced Salt Solution (HBSS), Calcium Chloride (10 mM stock solution), Phosphate Buffer Solution (1X PBS), MATRIGEL, 0.4% Trypan Blue solution and Trypsin.
  • FIGS. 27A-27C The same procedure was successfully performed on human liver tissue, as shown in FIGS. 27A-27C.
  • the MOSs were initially formed with about fifty cells, as shown in FIG. 27A.
  • day 18 in culture some of the MOSs showed cells having clusters and forming structures, while others had smaller structures or the cells did not divide.
  • Example 8 Cultured cell Micro-Organospheres
  • the MOSs may be formed from cell lines grown as part of a Patient Derived Xenograft (PDX).
  • PDX Patient Derived Xenograft
  • FIGS. 28A-28D illustrate MOSs formed from cultured PDX240 cells.
  • PDX240 cells are a Patient Derived
  • FIGS. 29A-29D show a similar experiment in which five PDX240 cells were initially included in each droplet forming each of the MOSs. With time in culture (e.g., from day 1 , day 3, day 5 and day 7, as shown in FIGS. 29A-29, respectively) the cells may divide and form structures.
  • the device may be otherwise oriented (rotated 90 degrees or at other orientations) and the spatially relative descriptors used herein interpreted accordingly.
  • the terms “upwardly”, “downwardly”, “vertical”, “horizontal” and the like are used herein for the purpose of explanation only unless specifically indicated otherwise.

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Abstract

La présente invention concerne un procédé pour tester des médicaments et des produits biologiques pour l' immuno-oncologie à l'aide de microorganosphères (MOS). Le procédé comprend la formation d'une pluralité de MOS à partir d'un tissu de biopsie de tumeur cancéreuse avec une telle formation consistant à dissocier des cellules à partir du tissu de biopsie de tumeur cancéreuse et à combiner les cellules dissociées avec un matériau de matrice fluide. La dissociation elle-même comprend un protocole de digestion enzymatique, le hachage du tissu de biopsie de tumeur cancéreuse, et l'incubation du tissu de biopsie de tumeur cancéreuse dans une solution de digestion avec agitation. Le procédé consiste également à tester au moins un médicament ou produit biologique pour l'immuno-oncologie à l'aide de la pluralité de MOS pendant 10 jours après la formation de la pluralité de MOS.
PCT/IB2022/058785 2021-09-16 2022-09-16 Procédés et appareil pour tester des médicaments et des produits biologiques pour l'immuno-oncologie à l'aide de microorganosphères WO2023042156A1 (fr)

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Publication number Priority date Publication date Assignee Title
WO2024054564A3 (fr) * 2022-09-07 2024-04-18 Xilis, Inc. Micro-organosphères liées au myélome multiple

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US20200032187A1 (en) * 2015-01-07 2020-01-30 Dana-Farber Cancer Institute, Inc. Microfluidic cell culture of patient-derived tumor cell spheroids
US20200232979A1 (en) * 2019-01-22 2020-07-23 Mayo Foundation For Medical Education And Research Microcapsules and Methods for Analyte Detection

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Publication number Priority date Publication date Assignee Title
US20200032187A1 (en) * 2015-01-07 2020-01-30 Dana-Farber Cancer Institute, Inc. Microfluidic cell culture of patient-derived tumor cell spheroids
US20200232979A1 (en) * 2019-01-22 2020-07-23 Mayo Foundation For Medical Education And Research Microcapsules and Methods for Analyte Detection

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2024054564A3 (fr) * 2022-09-07 2024-04-18 Xilis, Inc. Micro-organosphères liées au myélome multiple

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